Ignacio Pérez-Victoria

Largimycins are a recently discovered group of hybrid nonribosomal peptide-polyketide compounds belonging to the leinamycin family of natural products. They are produced in Streptomyces argillaceus by the lrg biosynthetic gene cluster, which includes three cytochrome P450 encoding genes. By characterizing the compounds accumulated by mutants lacking these three genes, we have determined their essential roles in largimycin biosynthesis. Specifically, LrgC2 is involved in converting an olefinic exomethylene group into an oxo (ketone) group at position C-9, while LrgC1 is responsible for forming an epoxide on the C-3 side chain. Also, it is proposed that LrgC3 acts at an early stage of the biosynthetic pathway, probably hydroxylating an alanine residue during peptide assembly. The chemical structure of the compounds produced by lrgC1- and lrgC2-minus mutants suggests that a hydrolytic dehalogenation step proceeds before the peptide-polyketide chain is off-loaded from the polyketide synthase LrgJ. Remarkably, some compounds generated by these mutants bear a C-3 side chain that is cyclized into a unique β-thiolactone ring, a structural feature not seen before in natural products. Furthermore, this work has revealed alternative ways of peptide-polyketide chain off-loading by macrocyclization, unveiling the unprecedented flexibility of the thioesterase domain of LrgJ for selecting different intramolecular nucleophiles.

Fungal genomes encode a huge biosynthetic potential to produce a wide diversity of chemical structures with valuable biological activities remaining silent or under-expressed with standard laboratory conditions. Fungal natural products provide numerous environmentally friendly properties that make them an attractive alternative to the use of synthetic pesticides for the control management of fungal diseases in plants. The main goal of this study was to explore the potential application of a library of natural products extracts from 232 diverse leaf litter associated fungi from South Africa for the discovery of new bio-fungicides. Fungal strains obtained from the MEDINA Fungal Collection were taxonomically classified following molecular and phylogenetic analyses, revealing a high diversity of fungal strains associated with leaf litter from endemic plants of South Africa. A previously designed library prepared following a specific OSMAC approach was employed in this work to evaluate the antifungal activity on a panel of four relevant fungal phytopathogens, using a recently developed HTS platform. The presence of the epigenetic modifier suberoylanilide hydroxamic acid (SAHA) during fungal fermentations in specific formulation media determined considerable changes in the metabolomic profiles that clearly influenced the diversity and quantity of bioactive metabolites, which also affected the activity hit-rates. As a proof of concept, we describe herein the discovery of the novel molecule libertamide upon the addition of SAHA during the fermentation of the strain Libertasomyces aloeticus CF-168990. Libertamide showed antifungal potential to control Zymoseptoria tritici, the causal agent of septoria leave blotch. As result of the study, we show that leaf litter from South Africa is still an untapped source of new fungal species with large biosynthetic potential to produce promising candidates for the discovery of new natural fungicides.

Fungal genomes encode a huge biosynthetic potential to produce a wide diversity of chemical structures with valuable biological activities remaining silent or under-expressed with standard laboratory conditions. Fungal natural products provide numerous environmentally friendly properties that make them an attractive alternative to the use of synthetic pesticides for the control management of fungal diseases in plants. The main goal of this study was to explore the potential application of a library of natural products extracts from 232 diverse leaf-litter-associated fungi from South Africa for the discovery of new bio-fungicides. Fungal strains obtained from the MEDINA Fungal Collection were taxonomically classified following molecular and phylogenetic analyses, revealing the richness and diversity of leaf-litter-associated fungi from endemic plants of South Africa. A previously designed library prepared following a specific OSMAC approach was employed in this work to evaluate the antifungal activity on a panel of four relevant fungal phytopathogens, using a recently developed HTS platform. The presence of SAHA during fungal fermentations in specific formulation media determined considerable changes in the metabolomic profiles that clearly influenced the diversity and quantity of bioactive metabolites, which also affected the activity hit-rates. As a proof of concept, we describe herein the discovery of the novel molecule libertamide upon the addition of SAHA during the fermentation of the strain Libertasomyces aloeticus CF-168990. Libertamide showed antifungal potential to control Zymoseptoria tritici, the causal agent of septoria leave blotch. As result of the study, we show that leaf-litter from South Africa is still an untapped source of new fungal species with large biosynthetic potential to produce promising candidates for the discovery of new natural fungicides.

To address the on‐going need for chemical novelty and the limited information on Planctomycetota secondary metabolism, we focused on exploring the recently isolated marine planctomycetal strain Alienimonas chondri LzC2 ^T to uncover its potential production of novel compounds. This work contemplates the description of a large‐scale cultivation study of strain LzC2 ^T , followed by metabolite extraction and compound isolation using chromatographic approaches, which resulted in the isolation of a novel molecule designated as alichondrichlorin. Structural elucidation of this new molecule was accomplished by a combination of high‐resolution mass spectrometry and nuclear magnetic resonance. The molecule was additionally screened for anti‐proliferative bioactivity against human tumoral and non‐tumoral cell lines. These cytotoxicity assays revealed a targeted effect of alichondrichlorin in the growth of tumoral cell lines, especially human breast adenocarcinoma MCF‐7 cell line (EC ₅₀ = 4.06 μM) without effect on the human non‐tumoral THLE‐2 cell line (EC ₅₀ > 50 μM).

A novel family of antibiotics, MDN-0057 to MDN-0060 (1-4), was isolated from liquid cultures of the fungus Ophiosphaerella korrae. These compounds incorporate two isocyanide groups in their complex structures that were elucidated by extensive spectroscopic analyses, including HRESIMS, and 1D and 2D NMR experiments. The relative configurations were determined by using J-based configuration analyses and the interpretation of key NOESY correlations consistent with the existence of a major conformation in solution. Mosher ester derivatization analysis allowed the establishment of their absolute configurations. All four compounds displayed in vitro antibacterial activity with a broad spectrum against Gram-negative pathogens.

Natural products play a crucial role in drug development, addressing the escalating microbial resistance to antibiotics and the treatment of emerging diseases. Progress in genome sequencing techniques, coupled with the development of bioinformatics tools and the exploration of uncharted habitats, has highlighted the biosynthetic potential of actinomycetes. By in silico screening for diazo-related gene genomes from twelve Streptomyces strains isolated from Attini leaf-cutting ants, the new crx biosynthetic gene cluster (BGC) was identified in Streptomyces sp. CS057. This cluster, highly conserved in several Streptomyces strains, contains genes related to diazo group formation and genes for the biosynthesis of 3,4-AHBA. By overexpressing the LuxR-like regulatory gene crxR1, we were able to activate the crx cluster, which encodes the biosynthesis of three 3,4-AHBA-derived compounds that we named crexazones (CRXs). The chemical structure of crexazones (CRXs) was determined by LC-DAD-HRMS-based dereplication and NMR spectroscopic analyses and was found to correspond to two known compounds, 3-acetamido-4-hydroxybenzoic acid (CRX1) and the phenoxazinone texazone (CRX3), and a novel 3,4-AHBA-containing compound herein designated as CRX2. Experimental proof linking the crx BGC to their encoded compounds was achieved by generating mutants in selected crx genes.

The development of efficient methods for dereplication has been critical in the re-emergence of the research in natural products as a source of drug leads. Current dereplication workflows rapidly identify already known bioactive secondary metabolites in the early stages of any drug discovery screening campaign based on natural extracts or enriched fractions. Two main factors have driven the evolution of natural products dereplication over the last decades. First, the availability of both commercial and public large databases of natural products containing the key annotations against which the biological and chemical data derived from the studied sample are searched for. Second, the considerable improvement achieved in analytical technologies (including instrumentation and software tools) employed to obtain robust and precise chemical information (particularly spectroscopic signatures) on the compounds present in the bioactive natural product samples. This chapter describes the main methods of dereplication, which rely on the combined use of large natural product databases and spectral libraries, alongside the information obtained from chromatographic, UV–Vis, MS, and NMR spectroscopic analyses of the samples of interest.

Bioprospecting the secondary metabolism of underexplored Actinomycetota taxa is a prolific route to uncover novel chemistry. In this work, we report the isolation, structure elucidation, and bioactivity screening of cellulamides A and B (1 and 2), two novel linear peptides obtained from the culture of the macroalga-associated Cellulosimicrobium funkei CT-R177. The host of this microorganism, the Chlorophyta Codium tomentosum, was collected in the northern Portuguese coast and, in the scope of a bioprospecting study focused on its associated actinobacterial community, strain CT-R177 was isolated, taxonomically identified, and screened for the production of antimicrobial and anticancer compounds. Dereplication of a crude extract of this strain using LC-HRMS(/MS) analysis unveiled a putative novel natural product, cellulamide A (1), that was isolated following mass spectrometry-guided fractionation. An additional analog, cellulamide B (2) was obtained during the chromatographic process and chemically characterized. The chemical structures of the novel linear peptides, including their absolute configurations, were elucidated using a combination of HRMS, 1D/2D NMR spectroscopy, and Marfey’s analysis. Cellulamide A (1) was subjected to a set of bioactivity screenings, but no significant biological activity was observed. The cellulamides represent the first family of natural products reported from the Actinomycetota genus Cellulosimicrobium, showcasing not only the potential of less-explored taxa but also of host-associated marine strains for novel chemistry discovery.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house “Carlos Sialer (CS)” strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide–depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.